Understanding the structural basis for the interaction of apoE with the LDL receptor (LDLR) will clarify hove the diverse array of proteins in this receptor family recognize ligands and will aid in the design of receptor mimics with therapeutic potential. The aims of this proposal are to: i) determine the contribution of specific LDLR residues to ligand binding by testing the hypothesis that electrostatic interactions are the primary means by which LDLR and apoE interact, ii) create a lipid-free multivalent ligand for the LDLR to examine the hypothesis that high affinity interaction arises primarily due to the presentation of multiple copies of apoE receptor binding domain, independent of significant structural alterations, and iii) discern the mechanism by which the epidermal growth factor precursor (EGFP) domain triggers acid- dependent ligand release. Binding will be initially analyzed using a solid phase binding assay, then a method such as fluorescence energy transfer or surface plasmon resonance will be developed for a more quantitative analysis of binding. Point mutations will be introduced into a minimal length LDLR to investigate the importance of charged residues, calcium coordination, and an exposed hydrophobic surface for ligand binding. The requirement for ligand multivalency will be investigated by expressing recombinant fusion proteins that consist of the N-terminal domain of apoE connected to coiled coil domains of defined oligomeric state. Acid-dependent ligand release will be explored by monitoring the effects of pH on the ligand binding affinity of LDLR constructs containing mutations of key residues in the EGFP domain.